Recent studies have shown crucial roles of fibroblast growth factor-23 (FGF-23) and klotho in regulating calcium and phosphate homeostasis. FGF-23 is an important in vivo regulator of phosphate homeostasis, while klotho is involved in regulating calcium homeostasis by interacting with the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5). Interestingly, Fgf-23 and klotho ablated (kl/kl, klotho-/-) mice have very similar physical, biochemical and morphological phenotypes;in vivo genetic manipulation of Fgf- 23 and klotho resulted in altered mineral ion homeostasis in these mutant mice. It is, however, not yet known how FGF-23, klotho and vitamin-D coordinately regulate mineral ion homeostasis. In this grant application we plan to use Fgf-23-/- , Fgf-23-/-1 a-hydroxylase mutant mice, and klotho-ablated and transgenic (Tg) mice as in vivo model systems to study possible in vivo interactions of these molecules, and their coordinated effects on mineral ion homeostasis. We propose to examine the effects of exogenous and endogenous FGF-23 on the induction of klotho in Fgf-23V and Fgf-23 -/-1 a-hydroxylase mice, and whether such interaction is a vitamin-D dependent process (Specific Aims 1 and 2). Our preliminary results suggest an in vivo interaction of FGF-23 and klotho. Furthermore, we found decreased renal expression of klotho in Fgf-23 mice;whether such decreased expression of klotho is partly responsible for premature aging-like features, and abnormal mineral ion homeostasis in Fgf-23 mice is not known. Therefore, we will determine the effects of restoration of klotho levels in Fgf-23-/- mice by generating Fgf-23-/-klotho-Tg mutant mice (Specific Aim 3). Finally to determine klotho -dependent and -independent functions of FGF-23, we would like to study Fgf- 23 -/-/klotho-/- mice, and Fgf-23 transgenic/klotho-/- mice. The long-term objective of this grant proposal is to determine in vivo function, and regulation of FGF-23, and klotho in physiological and pathological conditions. As a preliminary step of obtaining such objectives, we have recently generated and partially characterized both Fgf-23-/- , and Fgf-23 -/-1 a-hydroxylase double mutant mice. To study the effects of circulating FGF-23 on the induction of klotho to rescue the abnormal phenotypes of Fgf-23-/- , we generated another mouse model that is completely ablated for endogenous Fgf-23, but expresses FGF-23 in osteoblasts which is then released into circulation (Fgf-23-/-/Col1-FGF-23Tg mice). We will analyze the interrelationship of FGF-23, klotho and vitamin-D, and their effects on essential renal calcium and phosphate regulating molecules, including TRPV5, NaPi2a, Calbindin28k, NCX1, and PMCAbl and their effect on skeletogenesis. Successful completion of this proposed grant application would generate data that will form the basis to design strategies to manipulate abnormal mineral ion homeostasis and defective skeletal mineralization in wide range of diseases including rickets/osteomalacia, tumoral calcinosis and chronic renal failure, by developing novel therapies and fine tuning of the existing therapeutic modalities